1. Field of the Invention
This invention is directed to the field of laboratory apparatus and methods and particularly to laboratory vessels and methods for enabling economical establishment and maintenance of optimum environmental conditions for growing microbial cultures in either aerobic or anaerobic systems.
2. Prior Art
One of the most useful laboratory procedures in biological research is the growing of sample cultures of microbes in small vessels. It has long been common to grow such cultures in conventional erlenmeyer flasks, the cultures being placed, or inoculated, into a liquid medium hospitable to their growth, the flasks being plugged with some sort of porous material such as cotton, gauze, or foam.
Microbial cultures, depending on their type, may require aerobic or anaerobic environments.
It is critical to microbial cultures requiring aerobic environments that they receive sufficient oxygen. A paucity of oxygen is detrimental to optimum growth of the cultures, due to the resultant inhibition of the metabolic activity directly related to the oxidative process of the microbes. Research further indicates that lack of oxygen is also detrimental to the integrity and longevity of cells comprising the culture.
Microbial cultures derive their oxygen from that supply of oxygen which is dissolved in the liquid medium in which they are located. The conventional erlenmeyer flask is incapable of allowing sufficient oxygen transfer into the medium to satisfy the requirements of many types of microbes.
A liquid medium in a conventional erlenmeyer flask receives oxygen by means of transfer of oxygen into the medium from above the surface of the medium in the flask. The rate of transfer is directly related to the surface area of the air-medium interface. Since the surface area of the medium in a flask does not increase with increasing volume of medium, (indeed, it decreases in the case of flasks which narrow toward their tops) it becomes more difficult to maintain a sufficiently high oxygen level in a medium as its volume rises. Therefore, in the conventional flask, the amount of medium which can be used, and consequently the size of the culture that can be grown, are limited because the supply of oxygen that can reach the growing cells becomes more limited as the liquid volume within the flask is increased.
It is one object of this invention to provide for growth of microbial cultures in which ample oxygen can be transferred to the medium regardless of the surface-to-volume ratio of the medium.
Another limitation in the conventional methods of growing cultures is that the plugs used in the flasks, although permeable to the air, do limit the entry of air to the flask. It is a purpose of this invention to provide a method in which the entry of oxygen into a flask is not dependent upon its passage through conventional porous plugs.
It has been found that, in connection with the use of erlenmeyer flasks, greater oxygen transfer can be obtained by shaking the flasks by means of mechanical shaking machines. These shaking machines are frequently run, in practice, at standard speeds, and often such speeds are not appropriate to provide optimum oxygen transfer. Even where the machine is capable of very rapid shaking (which, on first examination, should yield high oxygen transfer) the efficiency of oxygen transfer is adversely affected by strong vortexing of the liquid medium which takes place in the vessels.
To correct this problem, and to further enhance oxygen transfer in shaken vessels, it is known to add baffles to the interior of the flasks to increase agitation of the liquid medium during shaking, and to reduce vortexing. Difficulties arise with theis technique, however, in the case of culturing of microbes which tend to form filamentous chains. This type of organism, when agitated in a flask having baffles, will tend to grow on the baffles themselves, or on the walls of the flasks, rather than in the medium itself, where optimum growth is obtainable.
A further purpose of this invention is to enable the introduction of abundant oxygen into the growth medium without the use of baffles. Additionally, it is a purpose to enable the introduction of abundant oxygen while lessening and sometimes eliminating the need for shaking of the flask.
Another problem with the conventional cotton, gauze, or foam plugs often used in the tops of erlenmeyer flasks is that these closures, by virtue of their ability to allow air to enter, and, especially at high temperature incubations, will also allow extensive evaporation of the medium and undesirable change in the volume thereof, upsetting the uniformity of the environment, and the quantitation of the contents of the flask.
Microbial cultures growing in a medium emit metabolic products some of which are gaseous or volatile. It is often desirable to analyze quantitatively these products of metabolism. Obviously, the porous plugs commonly used allow these products of metabolism to escape, making their measurement and analysis impossible. Another disadvantage of the use of porous plugs is that it is impossible, without the use of extremely elaborate and expensive laboratory apparatus, to determine with any degree of accuracy the amount of oxygen transferred to the medium and to the microbial culture.
It is a further purpose of this invention to obtain accurate estimation of oxygen absorbed by the microbial culture and quantitative determination of all metabolic products of the culture.
It is another object of this invention to provide an adaptable and relatively inexpensive method to permit variable control of aeration conditions so as to make such conditions optimal for the desired growth, metabolic activity, or viability of a given microorganism.
In prior art flasks, due to their incapability of air-tight sealing, the mechanical pressure of the gaseous phase of the microbial system cannot be regulated. Consequently, such flasks are of little or no utility in aerobic experiments in which oxygen tension is expected to function as a growth limiting factor.
Because of their "loose" closures, sterility in the prior art flasks can be maintained only fortuitously. The flask of this invention offers easy maintenance of internal sterility.
It is another object of this invention to provide an efficient and economical method to permit precise control over environmental conditions of a microbial culture for relatively long periods of time. To this end, this invention provides a laboratory flask with respect to which gaseous or liquid substances can be inserted and removed without substantially disturbing the environmental conditions therein, thus maintaining the stability and balance of the system, while still allowing for controlled changes of variables of the environment and sampling of substances within the flask.
It is still a further purpose of this invention to provide a laboratory method for the efficient and economical maintenance of precisely controlled environmental conditions for growth of microbial systems, said method adaptable to provide both aerobic and anaerobic environments.
The defects of the erlenmeyer flasks referred to above have been recognized by those skilled in the art for some time. Consequently, numerous modifications of the erlenmeyer flask and, indeed, other types of flasks, have been proposed in attempts to overcome these defects. However, all the prior art flasks known to applicant have one or more of the defects referred to above and only the flask and method of this invention successfully attains all of the desired advantages discussed above.